Lentiviruses belong to a genus of viruses of the Retroviridae family, and are characterised by a long incubation period. Lentiviruses can deliver a significant amount of viral RNA into the DNA of the host cell and have the unique ability among retroviruses of being able to infect non-dividing cells, so they are one of the most efficient methods of a gene delivery vector.
Lentiviral vectors, especially those derived from HIV-1, are widely studied and frequently used vectors. The evolution of the lentiviral vectors backbone and the ability of viruses to deliver recombinant DNA molecules (transgenes) into target cells have led to their use in many applications. Two possible applications of viral vectors include restoration of functional genes in genetic therapy and in vitro recombinant protein production.
Pseudotyping is the process of producing viruses or viral vectors in combination with foreign viral envelope proteins. As such, the foreign viral envelope proteins can be used to alter host tropism or an increased/decreased stability of the virus particles. For example, pseudotyping allows one to specify the character of the envelope proteins. A frequently used protein is the glycoprotein G of the Vesicular stomatitis virus (VSV), short VSV-G.
Efficient and controllable retroviral expression of a transgene is understood to require the presence of intron sequences. However, incorporation of such introns into retroviral vectors involves elaborate and time-consuming methods owing to the multi-step processes employed.
To date, viral gene transfer agents have not been useful for the treatment of diseases, without the transduction of stem cell populations, because of the host adaptive immune response, which prevents successful repeat administration.
Moreover, gene transfer to the airway epithelium has proven more difficult than originally anticipated. For example, the use of lentiviral pseudotypes that require disruption of epithelial integrity to transduce the airways, for example by the use of detergents such as lysophosphatidylcholine or ethylene glycol bis(2-aminoethyl ether)-N,N,N′N′-tetraacetic acid, has been linked to an increased risk of sepsis.
One example of a clinical setting which would benefit from gene transfer to the airway epithelium is treatment of Cystic Fibrosis (CF). CF is a fatal genetic disorder caused by mutations in the CF transmembrane conductance regulator (CFTR) gene, which acts as a chloride channel in airway epithelial cells. CF is characterised by recurrent chest infections, increased airway secretions, and eventually respiratory failure. In the UK, the current median age at death is ˜25 years. For most genotypes, there are no treatments targeting the basic defect; current treatments for symptomatic relief require hours of self-administered therapy daily. Gene therapy, unlike small molecule drugs, is independent of CFTR mutational class and is thus applicable to all affected CF individuals. However, to date no viral vector has met the requirements for clinical use, and the same applies to other diseases, particularly many other respiratory tract diseases.
In this regard, at least three major problems have been encountered. Gene transfer efficiency is generally poor, at least in part because the respective receptors for many viral vectors appear to be predominantly localised to the basolateral surface of the airway epithelium. Second, penetration of the respiratory tract mucus layer is generally poor. Finally, the ability to administer viral vectors repeatedly, mandatory for the life-long treatment of a self-renewing epithelium, is limited.
Administration of the vectors for clinical application is another pertinent factor. Therefore, viral stability through use of clinically relevant devices (e.g. bronchoscope and nebuliser) must be maintained for treatment efficacy.
Another example of a potential target for gene therapy is α1-antitrypsin (A1AT) deficiency. A1AT deficiency is an inherited disorder that may cause lung disease and liver disease. Symptoms include shortness of breath/wheezing, reduced ability to exercise, weight loss, recurring respiratory infections, fatigue and rapid heartbeat upon standing. Affected individuals often develop emphysema. About 10-15% percent of patients with A1AT deficiency develop liver disease. Individuals with A1AT deficiency are also at risk of developing a hepatocellular carcinoma.
A1AT is a secreted protein, produced mainly in the liver and then trafficked to the lung, with smaller amounts also being produced in the lung itself. The main function of A1AT is to bind and neutralise neutrophil elastase. A1AT gene therapy is likely to be of therapeutic value in patients with A1AT deficiency, CF and chronic obstructive pulmonary disease (COPD), where increasing or introducing A1AT may improve lung function.
A1AT therapy is also potentially valuable for the treatment of non-respiratory/non-pulmonary diseases, such as type 1 and type 2 diabetes, acute myocardial infarction, rheumatoid arthritis, inflammatory bowel disease, transplant rejection, graft versus host (GvH) disease, multiple sclerosis and infections, particularly viral infections, due to the effect of A1AT deficiency on other tissues/organs, such as the liver and pancreas (see, for example, Lewis Mol. Med. 2012; 18:957-970, which is herein incorporated by reference).
A1AT deficiency is an attractive target disease for gene therapy because the therapeutic threshold levels are well defined. A comparison of A1AT levels in subjects with the risk of developing emphysema/COPD determined a protective threshold level of 11 μM in serum, with levels below 11 μM are used as threshold for initiating protein augmentation therapy where available. A1AT levels in airway lining fluid are only ˜10% of serum level, because the lung epithelium constitutes a barrier and the therapeutic threshold in airway surface lining fluid is therefore considered to be 1.1 μM (see Ferraroti et al. Thorax. 2012 August; 67(8):669-74 and Abusriwil & Stockley 2006 Current Opinion in Pulmonary Medicine 12:125-131, each of which is herein incorporated by reference).
Six FDA-approved commercial formulations of A1AT protein isolated from pooled human blood are in clinical use in the US for the treatment of patients with severe A1AT deficiency (via weekly intravenous injections). Enzyme replacement therapy (ERT) is expensive ($100,000/year) and although biochemical efficacy for ERT protein augmentation therapy has been proven clinical efficacy has been more difficult to prove.
A1AT ERT is currently not accessible in all countries and currently not available in the UK. In addition, it is difficult to achieve sufficiently sustained tissue levels using current therapies, which may in part be responsible for the modest clinical efficacy observed so far.
Other attractive targets for gene therapy include cardiovascular diseases and blood disorders, particularly blood clotting deficiencies such as Haemophilia (A and B), von Willebrand disease and Factor VII deficiency.
Haemophilia, particularly Haemophilia A, is an attractive target for gene therapy. Haemophilia A is an inherited bleeding disorder caused by a deficiency or mutation of Factor VIII (FVIII). Its inheritance is sex-linked, with almost all patients being male. Bleeding is typically into the joints. Bleeding into the muscle, mucosal tissue and central nervous system (CNS) is uncommon but can occur. Disease severity is inversely proportional to the level of FVIII: less than 1% (<0.01 IU/ml) results in severe disease, with bleeding after minimal injury; between 1-5% (0.01 IU/ml-0.05 IU/ml) causes moderate disease, with bleeding after mild injury; and greater than 5% (>0.05 IU/ml) causes mild disease, with bleeding only after significant trauma or surgery.
There is accordingly a need for a gene therapy vector that is able to circumvent one or more of the problems described above.